Changes in modem agriculture have been shown to have detrimental affects on those bird species whose populations mainly inhabit the wider countryside. Recently extensive changes to the farmed landscape have occurred through implementation of European agricultural policy much of which has been concerned with decreasing agricultural production. Lapwing (Vanellus vanellus), with almost the entire British population breeding on farmland, is a species that is thought to have been particularly affected by agricultural change. A study attempting to assess the impact that modern, intensive arable farming had on a breeding population of lapwing was undertaken from 1995 to 1997 in south-west Lancashire. The study site had an open character and was predominantly winter farmed (cultivation occurring September-February)although more than a third of the area was spring cultivated. Permanent pasture and rotational set-aside (RSA) accounted for about 2% and 5% of the cropped area respectively. Lapwings preferred to nest in spring farmed fields and RSA. The overall nest survival was 55.5% and did not differ significantly between years. Hatching success was highest in RSA (88%), lowest in spring farmed fields (48.4%) and intermediate in winter farmed fields (57.2%). Hatching success was significantly higher in spring farmed fields than in other habitats but did not differ significantly between winter and spring farmed fields. The hatching success of winter farmed fields was elevated by the success of nests of fields cultivated late in the winter. The main cause of nest losses was farming operations which caused 77.4% of all nests lost. Predation accounted for 14.4% of nest losses and desertion for 8.2%. There were annual differences in nest destruction in winter farmed crops caused by differences in the timing of agricultural operations. Nest destruction was highest in unsown fields but was compensated for by a high rate of replacement and high hatching success in replacement clutches. Two types of crop were spring farmed, combinable (mainly cereals) and vegetable (mainly potatoes) crops. Higher hatching 2 success in spring cereals than in vegetables was related to the differing intensities of management between the two crop categories. Lapwing preferred to nest in dense aggregations and far from ditches both of which reduced the risk of predation. Low densities of nest predators were maintained in the study area by predator control. Chicks from larger eggs survived better than chicks from smaller eggs. Chicks undertaking movements from natal to rearing fields suffered high levels of mortality. Chick mortality was caused by poor body condition, entrapment in field boundary ditches or predation or from the interaction of these factors. Brood movements were influenced by the distribution of crop types. Spring farmed fields and pasture were used for rearing broods. Predation was the main proximate cause of mortality for radio-marked chicks and accounted for 52% of all losses. Predation was a significant mortality factor until chicks were at least 20 days old, whereas poor body condition (31 % of all radio-marked chick losses) and ditch entrapment (17% of all losses) only killed very young chicks. Fledging success or hatching success is thought to be the main limit on productivity. Dietary studies revealed chicks ate mainly beetles but their diet varied depending upon rearing location. Surface living chick prey was abundant throughout the season in arable fields and late-hatched chicks suffered higher mortality than those hatched earlier mainly due to an increase in predation late in the season. Recommendations to maintain or improve the conditions for breeding lapwing within the study site are discussed. They include proposals which could operate under the agrienvironment regulations of the CAP, such as an increase in the area spring farmed with the cultivation of both cereal and vegetables and changes to the management of rotational set-aside to make such fields more suitable for brood rearing.

No description available.

Circadian rhythms, i.e. rhythms exhibiting a cyclic behaviour with a period of approximately 24 hours, are present in the metabolism of most living organisms. The transcriptional processes, i.e. the processes associated with mRNA synthesis, critically contribute to their origination, and are responsible for most of the mechanisms which regulate gene expression levels in cells. Inhibition or activation of a putative transcriptionally regulated ‘child’ gene can be achieved via binding of proteins called transcription factors (TFs) to the gene promoter, a region of the DNA containing protein-specific binding sites. In this work, we investigate modelling and inference approaches for different scenarios of circadian transcriptional regulation. We focus on a system which comprises two transcription factors and a regulated child gene. We first perform parameter inference in the context of state-space models on simulated data from a mechanistic stochastic model describing this scenario. Additionally, we investigate the effect of data aggregation across different cells, and derive the smoothing equations for a destructive sampling scenario. In the second part of this work, we consider a situation in which an important regulator of a child gene has not been observed. We apply our model to mRNA expression levels of a subset of circadian genes of the Arabidopsis Thaliana model plant. Inference is in this case aimed at estimating both the model parameters and the unobserved transcription factor profile. We compare a posteriori the inferred transcription factor profiles with available time-series data for one important circadian regulator in the Arabidopsis Thaliana, namely late elongated hypocotyl (LHY), and identify similarities for a several genes known to belong to the central clock. Finally, we focus on a scenario of transcriptional regulation which includes an auto-regulatory negative feedback loop. This modelling framework is motivated by the availability of spatio-temporal imaging data of genes belonging to the mammalian central clock in mice suprachiasmatic nucleus (SCN), and in particular here we focus on Cry1. We introduce a distributed delay to account for nuclear export, translation, protein complex formation, and nuclear import, of the molecular species involved. To perform inference, we develop a novel filtering algorithm that can be applied to any system with distributed delays. We finally apply the methodology to Cry-luc spatio-temporal data, and find that parameter estimates are spatially distributed, with a marked difference between central and peripheral SCN regions.

The objective of the work described in this thesis was to develop novel experimental and analytical methods to study samples of anisotropically oriented fluorophores. The primary development was the fluorescence detection method for linear dichroism spectroscopy (FDLD). The experimental configuration of the measurement has the detector placed facing the propagation direction of the exciting incident light. Long-pass edge filters, selected so that their cut-off wavelength lies between the excitation and emission maxima of the sample’s fluorophore, were placed between the sample and detector to block transmitted incident light, whilst allowing Stoke’s shifted fluorescence emissions to pass freely. The experiment was designed to be conducted using a commercial circular dichroism (CD) spectropolarimeter, which has been adapted to measure linear dichroism (LD). A theoretical framework to calculate FDLD spectra using the output of such an instrument is presented, and used to generate the FDLD spectra of small molecules oriented on stretched oxidised polyethylene films and biological samples oriented in Couette flow. It was found that much of the information that can be obtained from LD may also be derived from FDLD spectra, however, FDLD possessed two clear advantages: i) fluorescence detection is highly sensitive, which significantly lowered the sample volume requirement of a measurement, and ii) fluorescence detection is more selective than absorption methods, as only chromophores that are also fluorophores are detected. A method for the cloning, expression and purification of the Escherichia coli actin homologue MreB is also presented. The composition of secondary structure elements within the protein obtained was analysed using CD and found to be in good agreement with literature values, taken from the crystal structure of Caulobacter crescentus MreB. Fluorescence spectra of the protein were recorded, which indicated that it may be possible to study its in vitro polymerisation kinetics using FDLD. Unfortunately, we were unable to obtain our Escherichia coli MreB in a sufficient yield to develop this assay. Finally, a novel method for detecting the wall shear stress (WSS) exerted on a specific point of a surface by a fluid flowing over it is reported. This information was derived from the response to a shear flow of a fluorescently labelled particle of M13 bacteriophage, which was tethered to the surface. The focus of this thesis was primarily on the analysis of the fluorescence signals. Using a custom made algorithm, microscopy images of the tethered phage particle were segmented to define the region of each image occupied by the particle, and to calculate its orientation and length at all time points. These data were used to calculate the WSS at the point of the particle’s attachment, and show that it varied when the surface was not uniform — highlighting a potential problem with commonly used methods for determining WSS that average over surface dynamics, such as particle image velocimetry and particle tracking velocimetry.

Female-female competition has been relatively overlooked in favour of male-male competition for mates, but it can also have important reproductive consequences. There are an increasing number of studies describing conditions where females compete to obtain breeding rank, gain access to or control resources or actively defend young. Communally breeding females are thought to be relatively egalitarian, sharing the cost of parental care with other females. Hence little attention has been paid to the potential for competition in such breeding systems, despite evidence of aggression and reproductive suppression between females. This thesis therefore explores the extent of competitive behaviours between female wild house mice (Mus musculus domesticus), a species with communal care of young, and investigates the physiological effects of competition and its consequences for breeding success and reproductive output. I examined the effects of age and other characteristics that may predict the degree of female competition. I identified that body mass, relative age of social partners, urinary testosterone concentration and reproductive experience were all useful predictors for the amount of competitive behaviour observed between female pairs. Following competitive female interaction I found that urinary testosterone and protein output increased, but there was no significant change in body mass and no significant effect on oestrus cycle length. Older females (> 12 months) with competitive experience had larger adrenal glands compared to females previously housed with their sisters, suggesting a possible stress response to competitive interaction. There was also evidence that competitively housed females had enlarged clitoral glands, which may play a role in signalling social status. As female house mice were found to compete and assume social ranks, I investigated the impact of female social status on male mate choice and mating behaviour. There was no evidence of significant male preference for more or less competitive females prior to or after competitive interaction in a choice test with restricted access to females or when presented with female odours. To investigate breeding behaviour I introduced female pairs to a male in semi-naturalistic enclosures, filming continuously over a four day period to examine mating attempts and female behaviour. Interestingly males mounted less competitive females either exclusively or preferentially during the test, with a small number of competitive females interrupting mating behaviour between their social partners and the male. Therefore males may prefer female partners that are less likely to act aggressively towards their advances. The effect of female competition on reproductive success was examined by comparing breeding success of subjects under solitary and communal breeding conditions. Despite the prediction that reproductive success increases for secondary litters in house mice, reproductive output was significantly reduced for more and less competitive females in the communal nest compared to previous output in a solitary nest. This finding illustrates the negative impact of competition on reproductive success. Females that gave birth first in communal nests also had significantly fewer pups present on post natal day one compared to females that gave birth second. Interestingly female offspring of more competitive females in this experiment went on to produce larger litters on average than females born to less competitive females. Litters were also likely to be male biased if females had been reared in a competitive environment rather than a solitary nest, suggesting that competitive ability and rearing environments can both influence reproductive success for offspring. These results, together with evidence in the literature, suggest that competition does occur between communally breeding females, and that reproductive success can be affected as a result. However competition between communal females may be less intense than between females in cooperative systems, where reproductive skew is biased towards one or two individual females in a group. Using a comparative analysis I found that cooperatively breeding species had increased reproductive output compared to other polytocous species, which is likely to be influenced by the presence of non-breeding helpers in the nest site. Cooperative species also had decreased inter-litter intervals compared to non-cooperative species, as well as a reduction in lactation length and protein content of milk. Communal species were found to have increased offspring growth and reduced sexual size dimorphism, suggesting that competition between females may have resulted in selection for increased female body mass. Together these results illustrate the significance of female competition in wild house mice, with consequences for mating behaviour and reproductive success, as well as the evolutionary implications of female-female competition in mammalian species residing in communal breeding systems.

The concept of cellular senescence is based on the notion that proliferation of normal diploid cells can only occur for a limited period of time after which cells slowly cease to divide and die. This limitation of lifespan for in vitro cell cultures was first described by Leonard Hayflick in 1961, going against the theory formulated by Alexis Carrel claiming that cells kept in culture have an unlimited potential for division (Carrel and Ebeling, 1921). Evidence from cell senescence studies have been used to explain the basis of healthy ageing as well as develop food supplements promoting the delay of ageing. Since these discoveries, there has been marked expansion of anti-stress gene response research with relation to the role of the transcription factor NF-E2-related factor-2 as well as healthy ageing with regards to dietary activators intake. This study examined the effects of sulforaphane (SFN) and hesperetin (HESP) on MRC-5 cell senescence in vitro as well as characterized gene expression and cell metabolism in cells escaping senescence by treatment with SFN and HESP and the mechanism of decrease glucose metabolism by SFN and HESP action linked to delay of fibroblast senescence. The effects of treatment of MRC-5 with the dietary bioactivators SFN and HESP was studied and shown to delay cellular senescence in these cells in vitro. Similar findings for SFN treatment of BJ cells were found previously by studies of the host team. Caloric restriction mimetic mechanisms by treatment with 1 μM SFN and 5 μM HESP was shown due to the decrease in culture glucose consumption increase with senescence in these treatment groups and this CR restriction mimetic effect and decrease in the flux of formation of D- and L-lactate in the SFN-treated group was consistent with previous studies done in CR and ageing in mice models (Hargopan, Ramsey and Weindruch, 2003). Moreover, in this study, SFN was shown to act as a CRM dietary bioactive through cellular contents of glycolytic intermediates with SFN-treatment (Bensaad et al., 2006). Finally, the mechanism by which SFN induces delay of senescence through CR was shown to be due to the extraction of Mondo A from the cell nucleus to the cytoplasm.

The formation of embryoid bodies has long been utilized to initiate differentiation of embryonic stem cells in vitro. The embryoid body provides an effective means of recapitulating early stages during embryogenesis and formation of the three germ layers. Current methodology for embryoid formation is extensive but exhibits a lack of standardisation and coherence. Here is shown a 3D culure system for controlled embryonic stem cell aggregation via a non-cytotoxic cell surface modification and cell-cell cross-linking. Embryoid body formation was found to be a complex relationship between embryonic stem cell aggregation, proliferation, death, cluster agglomeration, extracellular matrix deposition and structural reorganisation. Engineered embryoid bodies formed more rapidly and were significantly larger than those in control samples. Embryoid body characterisation revealed a layered internal structure resulting from poor nutrient and gaseous diffusion and consequent core necrosis after ≥ 5 days in suspension culture. Immuno-labelling and PCR amplification analysis of Brachyury, Nestin, Gata-4 and Oct-4 showed differentiation of mesoderm, ectoderm and endoderm on the embryoid body surface and internal undifferentiated cells, respectively. Engineering appeared to enhance mesoderm differentiation, a progenitor of the osteogenic lineage. Embryoid bodies in settled culture spread outwards to form a plateau of collagen matrix which was later mineralized through differentiated osteoblast function. Quantification through Alizarin Red stained bone nodules and alkaline phosphatase activity demonstrated osteogenic differentiation enhancement within engineered samples. Dex-loaded poly-(lactic co-glycolic) acid polymer microparticles were found to be an effective method for delivery of osteo-inductive factors to internal undifferentiated embryonic stem cells within the embryoid bodies. These findings show that the proposed 3D culture system provides reliable and repeatable methodology for the controlled formation of embryoid bodies which exhibit enhanced osteogenic differentiation. It is hoped that these engineered embryoid bodies could be used to efficiently generate homogeneous bone tissue for clinical application.

On the Tibetan Plateau, there is a long history of animal farming practices. Although Tibetan pastoralism had been successful in the past to support the population, the problems of increasing demand and impact in the face of more scarce resources and global climate change are challenging pastoralists. The problem is even more pronounced in the Tibetan Plateau’s unique natural and socioeconomic conditions. Based on the perception of the problem, the Chinese government started a reform programme aiming at changing the nomadic practices in Tibet. Tibet today is in transition, not only in terms of pastoralism, but also that of culture, of institutions, and of economy, nevertheless the transition in pastoralism as a livelihood and source of income will have significant implications. The usefulness of the resilience concept in examining a complex system’s innovation, development, disturbance and reorganisation makes them suitable tools in the study of historical changes and the future of Tibet, as the area is under human management, and subject to the influence of changes in nature and external policies. In this study remote sensing and mathematical modelling approaches are used to assess ecological resilience in the region. The advantage of remote sensing allows the researcher to observe and analyse a large area as well as recent changes, and to examine the spatial pattern of these changes. The model simulates the dynamics of the grassland system given the current condition. The key functions linked to the system’s resilience can be examined in this model and provide information on the system’s sustainability. The simulation shows that the nomadic pastoralism system can better adapt to disturbances of known intensity and frequency than the sedentary style. However, the trend of climate change and population increase may require a change of organisation and practices for the system to be sustained.

Cytochrome family P450 4A encode a major group of enzymes which are involved in the mechanism of peroxisome proliferation in rodents. Induction of CYP4A expression by peroxisome proliferators is due to transcriptional activation, mediated via the peroxisome proliferator activated receptor alpha. Cyp4a enzymes catalyse the w-hydroxylation of fatty acids and eicosanoids, and it has been suggested they thereby play a pivotal role in blood pressure regulation. Murine Cyp4a10, Cyp4a14 and Cyp4a12 genes have been reported by Bell et al (1993) and Heng, et al (1997). There is contradictory evidence in the literature concerning the expression of lauric acid hydroxylase (LAH) and Cyp4a-related protein in mouse liver and kidney. We have previously shown that Cyp4a12 is expressed at high level in a male-specific fashion in liver and kidney of mouse (Bell et al 1993). However, various workers have reported the presence, or absence, of Cyp4a proteins, or LAH, in male mouse liver. Work by Hiratsuka et al (1996) demonstrate that ddY mice show a male specific expression of LAH and Cyp4a-related protein in the liver, while other strains Balb/c and C57BL/6 exhibit no sex difference in neither enzyme or protein. In the kidney, ddY, Balb/c and C57BL/6 all show sexual dimorphism in the expression of both LAH and Cyp4a related protein. The aim of this project was to characterise the expression of mouse Cyp4a12 and resolve the conflicting results in the literature. Findings demonstrate that there is a male specific expression of Cyp4a12 in male liver and kidney. The data differs from Hiratsuka et al in that their findings present no sex difference in Balb/c and C57BL/6 expression of Cyp4a proteins. Hiratsuka et al also determined that in the kidney of ddY, Balb/c and C57BL/6 there is no sex difference in expression of neither enzyme or protein. Data here agrees with these findings and suggests that the sexual dimorphism exhibited by the LAH and Cyp4a related protein in the kidney is due to constitutive expression of Cyp4a12. Thus it appears that there is till a discrepancy between the Cyp4a12 hepatic expression pattern presented here and that of the enzyme and protein determined by Hiratsuka et al. An explanation may infer that the LAH and Cyp4a-related protein measured by Hiratsuka et al is not only Cyp4a12 but also another member of the Cyp4a family. Further work is required to establish what Cyp4a is expressed predominately in the female. Work by Henderson et al (1994) supports the case that this Cyp4a candidate may be Cyp4a10. Continuing studies will clarify the expression pattern of the Cyp4a and also investigate the mechanism of regulation of the Cyp4a family genes and the expression of male specific genes.

Part One Preparations of nucleoprotein having reproducible characteristics were obtained from calf thymus glands by a mild extraction procedure. A preliminary examination of the dissociation of the complex was made and its physico-chemical behaviour was investigated in relation to that of its nueleic acid component. The nucleoprotein was highly aggregated in solution at low ionic strength, but at high ionic strength a weight-average molecular weight lower than that of nucleic acid itself provided additional evidence for the dissociation of the nucleoprotein. Spectrophotometric studies showed that the nucleoprotein was denatured by heat and by alkali in a similar manner to nucleic acid, although denaturation by heat was slightly retarded. The spectrophotometric titration behaviour of the two substances was almost identical, but their potentiometric titration curves differed significantly. Nucleoprotein solutions exhibited the features of nucleic acid devolving from its unique helical structure, indicating that this structure is retained by the nucleic acid within the complex. Part Two A general reaction has been discovered between amino sugars and aldoses for which the essential requirements are a free amino group and an aldehyde group. Three compounds (A, B and C) were separated by ion- exchange chromatography from a mixture obtained by heating D-mannose with D-glucosamine hydrochloride. Compound B was identified as 5-hydroxymethyl furfuraldehyde (HMF) from its ultra-violet absorption spectrum, chromatographic behaviour, and distribution coefficients between two different solvent systems, and was characterised as its 2:4-dinitrophenylhydrazone. Compound A was very unstable and was hydrolysed by cold water to mannose (characterised as its p-nitranilide) and glucosamine (characterised as its carbobenzoxy derivative). On heating in aqueous solution, the hydrolysis was accompanied by degradation to HMF (characterised in the same way as compound B), and the formation of melanoidins. The HMF isolated from the reaction mixture was shown to arise from compound A and C14-HMF was obtained when the condensation was effected with a mixture containing C-mannose and glucosamine hydrochloride, showing that the HMF originated in the mannose moiety of compound A. From these and other experiments, the structure N-mannosyl-glucosamine was assigned to compound A. Compound C was stable in aqueous solution, and hydrolysis with acid gave equimolecular quantities of mannose and glucosamine hydrochloride, but was not accompanied by a browning reaction. On the basis of periodate oxidation experiments, its behaviour in the Elson-Morgan reaction, and a number of other colour tests, compound C was provisionally assigned the structure 6-O-α-D-mannosyl-Z-amino-2-deoxy-D-glucose. Certain aldehydes were found to behave as bases on a sulphonated polystyrene resin. This interesting discovery may provide the basis of a new method for the separation and analysis of aldehyde mixtures.